The technique of fluorescence was first introduced by Coons in 1941. He used a blue fluorescing anthracene compound coupled to pneumococcus antiserum to detect bacterial antigens in tissue section. Subsequent to this initial discovery, many fluorescing materials have been investigated, but only two, the fluorochromes fluorescein and rhodamine, are widely used, particularly in the form of fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) respectively. FITC covalently binds to proteins at alkaline pH through the epsilon (.epsilon.) amino residues of lysine and through terminal amino groups. FITC's adsorption maximum is at 490-495 nm and it emits its characteristic green color at 517nm. TRITC likewise binds to proteins, has its absorption maximum at 541 nm and emits its characteristic red color at 572 nm.
Fluorescence is the emission of light of one wavelength (color) by a substance that is being irradiated by light of a different wavelength. The emitted light is always of lower energy, hence longer wavelength, then the incident light. In clinical use, the strength of the fluorescence is dependent on the efficiency with which the fluorochrome transforms incident light into emitted light, the amount of dye present in the specimen under observation and the intensity of the incident light. The dye known as Texas Red (sulforhodamine 101 sulfonyl chloride or sulforhodamine acid chloride) has previously been investigated for clinical use in conjugation with phycoerythrins, but major problems were encountered. These problems were low fluorescent efficiency, inadequate energy transfer from the phycoerythrin to Texas Red and the instability of the phycoerythrin-Texas Red conjugate. Phycoerythrin-Texas Red conjugates are desirable, however, because the overlap of their absorption and emission spectra have the potential to give a strong fluorescence signal.
Low fluorescent efficiency occurs whenever fluorescent chromophores are spatially adjacent to each other. It is usually called concentration quenching. See R. P. Hughland, "Excited States of Biopolymers", R. F. Steins, Ed., p 47 (Plenum Press, New York, 1983). However, high levels of labelling, resulting in chromophores being spatially adjacent to each other, are required in order to assure adequate energy (electron) transfer from the phycoerythrin to the acceptor dye chromophore. The net result is that the trade off required by the opposing effects results in less than optimal performance. Recently, A. N. Glazer et al. have covalently linked a phycoerythrin to an allophycocyanin to produce a highly fluorescent tandem conjugate with an energy transfer efficiency of 90%. See A. N. Glazer et al., T.I.B.S, 9:423 (1984); Biophysics J., 43,386-386 (1983); and U.S. Pat. No. 4,542,104 (See also U.S. Pat. No. 4,520,110 to L. Stryer et al. describing the use of phycobiliproteins as fluorescent probes for the analysis and separation of molecules and cells). However, forming a conjugate from two naturally occurring pigments derived from algae is much different from conjugating a synthetic dye such as Texas Red. In fact, the procedures usually followed for conjugating reactive dyes to proteins does not work with phycoerythrin-Texas Red. Using such procedures, one obtains a complex with a low energy transfer efficiency at low levels of labelling or fluorescence quenching at high levels of labelling. Texas Red forms a conjugate with a phycoerythrin by reaction of its sulfonyl or acid chloride moiety with an amine group of phycoerythrin or other phycobiliprotein
Phycobiliprotein/amine-reactive dye conjugates are known and some, for example, phycoerythrin-Texas Red conjugates, are commercially available. For example, the phycoerythrin-Texas Red conjugate known as DuoCHROME.TM. is available bound to streptavidin from Becton Dickinson Immunology Systems, Mountain View, Calif. (Catalog No. 9026). The available conjugates, however, suffer from the fact that they do not have a uniform phycoerythrin-Texas Red ratio throughout the individual conjugate members. There are present overlabelled and underlabelled species as well as species having the desired or optimum degree or range of labelling. Consequently, energy transfer/quenching problems can arise depending upon the distribution of labelled species within the entire sample.
This invention solves the energy transfer/quenching problem encountered in the preparation of phycobili-protein/amine-reactive conjugates in general by preferentially labelling sites close to the chromophore regions of a phycobiliprotein with an amine-reactive dye and separating overlabelled and underlabelled conjugates from conjugates having the desired degree of labelling by chromatographic methods; for example, by exploiting the differences in hydrophobic character of conjugates having different degrees of labelling.